Domica Cave is located in the Slovak Karst National Park in southern Slovakia. Heterotrophic cultivable psychrophilic and mesophilic microbiota were confirmed to be prevalent in this cave. Escherichia coli was the most abundant bacterium, followed by Enterobacter species and Serrratia species. Enterobacter cloacae isolates belong to the group of faecal contaminants (coliforms) of concern in water. Their status in cave ecosystem is questionable, but we observed E. cloacae in water samples all years round suggesting an autochthonous origin. We were concerned with possible contamination of the cave water with resistant E. cloacae from animal farms located 2-3 km from the cave. We tested 36 aquatic isolates of E. cloacae from Domica Cave to β-lactam resistance. The majority of tested isolates were resistant to more antibiotics (from 3 to 10), 5 isolates were resistant to more than 10 antibiotics, and 1 isolate was resistant to ampicillin. Resistance to the broad spectrum of β-lactams correlated with resistance mechanisms due to an expression of blaTEM, blaSHV and blaCTX-M genes. The majority of isolates possessed a combination of tested resistance genes. We assume a direct impact of long-term human agricultural activities in the area of the Domica Cave to the conserved microbiota of karst system.